POLYPLOIDS AND SEX DETERMINATION I N CAENORHABDITIS ELEGANS JAMES E. MADL AND ROBERT K. HERMAN
Department of Genetics and Cell Biology, Unizjersity of Minnesota, Saint Paul, Minnesota 55108 Manuscript received June 22, 1979 ABSTRACT
Tetraploid stocks of Caenorhabditis elegans var. Bristol carrying autosomal and X-linked markers have been produced. Tetraploid hermaphrodites fall into two categories: those that give about 1% male self-progeny and those that give 25 to 40% male self-progeny, The former are basically 4A;4X-four sets of autosomes and four sex chromosomes-and the latter are 4A;3X. Males are 4A;ZX. (Diploid hermaphrodites are 2A;2X; males are 2A;IX.) Triploids wsre produced by crossing tetraploid hermaphrodites and diploid males. Triploids of composition 3A;3X are hermaphrodites; 3A;ZX animals are fertile males. Different X-chromosome duplications were added to a 3A;ZX chromosome constitution to increase the X-to-autosome ratio. Based on the resulting sexual phenotypes, we conclude that there exists on the C . elegans X chromosome at least three (and perhaps many mare) dose-sensitive sites that act cumulatively in determining sex.
A tetraploid stock of the small, free-living nematode, Caenorhclbditis elegans, was obtained some years ago by NIGON (1949a, 1951a, 1951b) from a heat-treated culture and maintained for 78 generations until accidentally lost. NIGON ( 1949a) reported that tetraploid hermaphrodites were larger than diploids and fell into two classes, which he called thklygknes and allklogches, based on the sex ratios of their self-progeny. Thklygknes gave about 0.7% male progeny and alldogknes gave about 40%. NIGONalso inspected Feulgenstained oocytes and spermatocytes from tetraploid animals and decided that th4ygknes were basically 4A;4X (four sets of autosomes and four sex chromosomes), alldogknes were 4A;3X, and males were 4A;2X. He also noted that many meiocytes showed irregular chromosome constitutions and suggested that the numerous sterile and weakly fertile animals he encountered may have been aneuploid (NIGON 1951b) . NIGON (1949b) had earlier shown that diploid hermaphrodites were 2A;2X and males were 2A;IX. I n the work reported here, we produced tetraploid lines of C. elegans var. Bristol carrying X-linked and autosomal visible markers (BRENNER 1974) and used these lines in crosses with diploid male stocks, some of which carried X-chromosome duplications, to investigate the relationship between chromosome composition and sex determination in C. elegans. Genetxs 93: 393402 October, 1979
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J. E. M A D L ARTD R. K. H E R M A N MATERIALS AND METHODS
The mutations and X-chromosome duplications used, the methods of culturing the animals and the fluorescence staining procedures have been described by BRENNER(1974) and HERMAN, MADLand KARI(1979). Tetraploid hermaphrodites whose progeny were to be enumerated were transferred daily to fresh plates. Tetraploid progeny to be classified by progeny testing were picked singly as larvae to be sure that no mating had occurred. Our original tetraploid line was derived as follows. Separate cultures of N2 (wild type) males and dpy-11 V; unc-3 X hermaphrodites, normally grown at 20”, were shifted to 27.5” for 12 hr and then mated. Wild-type hermaphrodite progeny were picked and screened for unusually low frequencies of Dpy and Unc phenotypes among their self-progeny. One line of very low fertility segregated males and hermaphrodites that seemed larger than normal and showed, by fluorescence staining, to hace a chromosome count higher than diploid-probably triploid originally. Continued selection for large animals of higher fertility eventually produced a quite stable tetraploid line. This stock continued to segregate Dpy and Unc animals, and we established homozygous tetraploid stocks derived from it: SP344: dpy-11; unc-3 X; SP345: dpy-11 V; SP346: wild-type; and SP347: unc-3 X. SP347 was mated with dpy-5 I / + diploid males, from which wild-type triploid hermaphrodites were picked. Continued selection for higher fertility of Dpy Unc segregants eventually resulted in the SP34.3 tetraploid stock, dpy-5 I ; unc-3 X . SP348, which is dpy-11 V ; unc-6X, was established i n analogous fashion using SP345 and mnDp30(X;f)/unc-6/0 diploid males to generate the triploid intermediate. A Zeiss Universal microscope was used for Nomarski differential interference microscopy. RESULTS
Tetraploids: I n agreement with NIGON( 1951b) we found that tetraploid hermaphrodites were of two types, based on the frequencies of male self-progeny. Those that gave 10% or less male self-progeny we called LFM (low frequency male producer), and those that gave more than 10% male self-progeny we called HFM. Animals with less than five progeny were not classified. Table 1 gives the frequencies of self-progeny from LFM and HFM animals. The data show that HFM animals segregated not only a large proportion of males but also large fractions of both HFM and LFM animals, whereas LFM hermaphrodites gave predominantly LFM progeny. The frequencies in Table 1 agree well with NIGON’S data (1951b), with the possible exception of the category termed “not classifiable.” NIGONnoted that many tetraploid animals were sterile or weakly fertile, but he did not quote frequencies for that class. Wild-type and dpy-22 V ; unc-3 X tetraploids gave similar self-progeny ratios from LFM and HFM animals, and the data from both have been combined in Table 1. The egg-hatching ~
TABLE 1
Self-progeny of tetraploid hermaphrodites Parent
LFM
Progeny frequencies HFM Male
LFM HFM
0.75 0.15
0.06 0.30
0.01 0.26
Not classifiable.
Number of progeny counted
0.18 0.29
936 701
* These were animals, some arrested as larvae, that gave less than five self-progeny.
C.elegans POLYPLOIDS
395
Irequency from LFM hermaphrodites was measured: 87% of 218 eggs hatched. The average brood size of ten dpy-12; unc-3 LFM hermaphrodites was 65 and of five wild-type LFM hermaphrodites was 84. Diploid hermaphrodites generally give about 300 animals per brood. W e stained oocytes and a few male spermatocytes with Hoechst 33258, with results that were consistent with NIGON'S cytological findings; for example, the oocyte shown in Figure lb, showing 12 bivalents, is typical of those found in the progeny of LFM animals, but we have also observed irregular patterns in which one or two extra chromosomes were present or one or two chromosomes appeared to be missing. Triploids: Triploid animals were produced by mating diploid N 2 males and tetraploid hermaphrodites bearing an autosomal dpy marker and an X-linked unc. The oocytes of wild-type hermaphrodite progeny generally showed, by fluorescence staining, 18 chromosomes, often appearing as six bivalents and six univalents, as shown in Figure lc. Although we have not made measurements, triploid animals are clearly larger than diploids. Of 3,864 eggs laid by triploid hermaphrodites, only 15% hatched. This frequency is still much higher than would be expected if only euploid zygotes hatched, and indeed, of 187 adult progeny of triploids, 32% were hermaphrodites that gave four or fewer offspring and 17% were males. Furthermore, oocytes of progeny from triploids were often seen to be aneuploid. The results of mating diploid males with tetraploid hermaphrodites depended on whether the hermaphrodites were LFM or HEM. I n the case of LFM hermaphrodites, we would expect most ova to be diplo-X, half of which would be fertilized by X-bearing sperm to give wild-type 3A;3X hermaphrodites, and half of which would be fertilized by nullo-X sperm to give 3A;2X Unc-3 animals. Since the vast majority of the Unc-3 animals issuing from this cross were in fact male (Table 2), we conclude that 3A;2X individuals are male. The rare Unc-3 hermaphrodites and wild-type males could be due to the occurrence of triplo-X and haplo-X ova, respectively. When HFM animals were crossed with diploid males, many wild-type male progeny were produced (Table 2), presumably as the result of the fusion of unc-3 haplo-X ova and unc-3+ haplo-X sperm. That at least some of these males were in fact unc-3/unc-3+ was shown by mating them with dpy-21 V; unc-3 X diploid hermaphrodites: non-Dpy Unc-3 hermaphrodite progeny, which must have received an unc-3 gene from the triploid male, were produced. This result also showed that triploid males can be fertile. Another point to be made from
FIGURE1 .--Fluorescence microscopy of oocytes stained with Hoechst 33058. (a) Diploid. (b) Tetraploid. (c) Triploid. Magnification in a-c: 1760x.
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TABLE 2
Cross progeny of wild-type diploid males and tetraploid hermphrodites Hermaphrodite parent'
dpy-5 I ; unc-3X LFM dpy-11 V ; unc-3 X LFM dpy-11 V ; unc-6X LFM d p y - l i V ; unc-3 X HFM
Frequencies of non-Dpy phenotypes Wild-type Wild-type Unc-3 Unc-3 hermaphrodite male hermaphrodite male
0.52 0.51
0.55 0.35
0.02 0.04 0.03 0.39
0.02 0.02 0.01 0.00
0.M 0.43 0.41 0.26
Number of progeny counted
901 1,139 641) 298
* Each mating plate contained a single hermaphrodite. The phenotype of H F M animals used in crosses was confirmed on the basis of the frequency of males among (Dpy Unc) self-progeny. For LFM crosses, the progeny of LFM animals were used, and their classification as LFM was confirmed on the basis of the frequency of wild-type male cross-progeny.
the data of Table 2 is that only 26% of the cross progeny of the HFM animals were Unc-3 (as compared with 45% from LFM animals), which suggests that many nullo-X sperm were lost to inviable zygotes. The simplest interpretation of this result is that 3A;lX zygotes are inviable. We have also generated triploids by crossing tetraploid males and diploid hermaphrodites. We would expect these triploids to be largely 3A;2X, and indeed, 98 % of the cross progeny (among 295 animals counted) were male. Triploids bearing X-chromosome duplications: If sex is determined in C. elegans, as it is in Drosophila, by the X-to-autosome ratio, then a ratio of 0.67 (in triploids) gives rise to a male and a ratio of 0.75 (in tetraploids) gives a hermaphrodite. (In normal diploid males and hermaphrodites, the X-to-autosome ratios are 0.5 and 1.0, respectively.) We were therefore prompted to construct animals with intermediate X-to-autosome ratios by adding X-chromosome duplications to a 3A;ZX chromosome constitution. We used five different X-chromosome duplications, all of which carry unc-3f and one or more neighboring loci MADLand KARI (Figure 2 ) and are translocated to LGZ or LGZZ (HERMAN, 1979). Each of the five duplications is homozygous viable in diploid hermaphrodites, suggestifig that very little of the autosomal linkage group to which each i s attached is missing. We conducted two sets of experiments: in both sets, males of genotype Dp/+; unc-3/0 were crossed with LF'M tetraploid hermaphrodites homozygous f o r an autosomal d p y marker and an X-linked unc marker. In one set of experiments (Table 3 ) , the unc marker was unc-3, and in the other (Table 4),unc-6, which is not covered by any of the duplications, was used. The relative frequencies of the four sperm genotypes were determined from control crosses using diploid d p y ; unc-3 hermaphrodites; that is, in these crosses Dp/X, Dp/O, X and nullo-X sperm produced wild-type hermaphrodites, wild-type males, Unc non-Dpy hermaphrodites, and Unc non-Dpy males, respectively. The results of the crosses with diploid hermaphrodites are given in Table 3 and suggest a tendency for the Dp-bearing chromosome, particularly in the case of mnDpl0, to segregate from the X during meiosis in males (HERMAN. MADLand KARI 1979). In any case, if we compare the triploid progeny ratios in Table 3 with the diploids, we see that in three cases the frequency of wild-type triploid hermaphrodites increases
C . elegans POLYPLOIDS
---
dpy-6
~ n c - 8 4~ c - 3 unc-7
UnC-9
I
I
I
I
397
I
I
I
I
I
mnDp8 ( X ; I 1
mnDp9 ( X ; Z )
mnDp70 ( X ; l )
-
I
I
mnDp25 (41)
P
mnDp27
(&n1
FIGURE 2.-Genetic extents of the X-chromosome duplications used in this work (HERMAN, MADLand KARI 1979). TABLE 3 Cross progeny of X-duplication-bearingdiploid males and diploid or LFM tetraploid hermaphrodites
Duplication*
Ploidy of hermaphrodite+
mnDp8 mnDp8 mnDp9 mnDp9 mnDplO mnDplO mnDp25 mnDp25 mnDp27 mnDp27
diploid tetraploid diploid tetraploid diploid tetraploid diploid tetraploid diploid tetraploid
Frequencies of non-Dpv phenotypes Unc-3 Wild-type Wild-type Unc-3 male hermauhrudite male hermauhrodite
0.27 0.27 0.27 0.09 0.27 0.02 0.25 0.12 0.27 0.20
0.25 0.27 0.21 0.39 0.21 0.45 0.24 0.38 0.26 0.28
0.27 0.25 0.28 0.28 0.33 0.32 0.28 0.29 0.26 0.31
0.22 0.21 0.23 0.24 0.20 0.20 0.23 0.21 0.22
0.u)
Number of progeny counted
1,017 272
605 658 357 908 813 536 75 1 836
* Males all had the following genotype: Dp/+; unc-3/0.
tHermaphrodites were all homozygous for an
and unc-3 X .
autosomal dpy marker (dpy-5 I or dpy-11 V )
TABLE 4 Cross-progeny of Dp/+; unc-3/0 diploid males and dpy-I 1 V; unc-6 X LFM tetraploids
Duplication
mnDp8 mnDp9 mnDplO mnDp25 mnDp27
Frequencies of non-Dpy phenotypes Wild-type Wild-type Unc-6 hermaphrodite male hermaphrodite
0.56 0.53 0.53 0.56 0.54
0.04 0.02 0.03 0.05 0.03
0.02 0.17 0.29 0.12 0.02
Unc-6 male
Number of progeny counted
0.38 0.28 0.16 0.28 0.41
518 389 280 520 288
398
J . E. MADL AND R. K. HERMAN
at the expense of wild-type triploid males. This effect was most extreme in the case of mnDpl0, where very few wild-type males were produced, but it is also clear with mnDp9 and mnDp25. We conclude that adding mnDpZO to 3A;2X generally (if not always) gives a hermaphrodite, whereas adding mnDp9 or mnDp25 sometimes gives a hermaphrodite and sometimes a male. Indeed, in both the mnDp9 and mnDp25 crosses, about 10 to 20% of the animals classified in Table 3 as males, on the basis of male-like tails, proved upon closer inspection by Nomarski microscopy to be obvious intersexes, showing besides some development of a male-like tail, some oogenesis and sometimes even fertilized eggs and a vestigial vulva (Figure 3). Adding mnDp8 or mnDp27 to 3A;2X appeared to permit normal male development. Wild-type males appeared at about the same frequency as in diploid crosses, and intersexes were not common: 67 wild-type males and 67 non-Dpy Unc-3 males from a mnDp8 cross were inspected by Nomarski microscopy and no indication of intersexuality was found. As expected, the sex ratios for Unc progeny (which did not contain duplications) in every case were the same in triploids versus diploids in Table 3. I n the second set of experiments, in which the D p / + ; unc-3 X / O diploid males were crossed with individual dpy-11 V; unc-6 X LFM animals, the 3A;2X animals were represented by the non-Dpy Unc-6 progeny, roughly half of which (or more, particularly in the case of mnDpZ0; see diploid results in Table 3) should carry the duplication. If a duplication converts 3A;2X males to hermaphrodites, then the frequency of non-Dpy Unc-6 hermaphrodites, which is about 0.02 in the absence of a duplication (Table 2). should increase at the expense of the non-Dpy Unc-6 male class. Furthermore, the sex ratio of non-Dpy non-Unc animals (which are 3A;3X) should be unaffected by the duplications. These predictions were satisfied by the results, given in Table 4. Over half of the non-
FIGURE3.-Nomarski microscopy of three intersex aninials. (a) 3A;2X/mnDp25 with a male tail and oocytes. Magnification: 1 0 0 ~ (b) . 3A;2X/mnDp25 with a male tail and fertilized eggs. Magnification: 1 9 0 ~ .(c) 3A;2X/mnDp9 with a partially developed male tail and fertilized eggs. Magnification: 2 3 0 ~ .
C. elegans POLYPLOIDS
399
Dpy Unc-6 progeny in the case of the mnDplO cross were hermaphrodites. As in the first set of experiments, mnDp9 and mnDp25 appeared to be partially effective in converting 3A;2X animals to hermaphrodites, and, as before, many of the classified male progeny proved upon closer inspecticln to be intersexes. Also as before,nnDp8 and mnDp27 appeared to be ineffective in converting 3A;2X males to hermaphrodites. DISCUSSION
We can use the data of Table 2 to estimate the relative proportions of haplo-Xi, diplo-X and triplo-X ova produced by LFM (4A;4X) and HFM (4A;3X) animals, on the basis of the following assumptions: diploid males produce equal proportions of haplo-X and nullo-X sperm, 3A;jX is inviable, 3A;2X is male, and 3A;3X and 3A;4X are viable hermaphrodites. The rationale for assuming the viability of 3A;4X is simply that 2A;3X animals are viable (HODGKIN, HORVITZ and BRENNER 1979), but the calculations are very insensitive to this assumption in any case. We calculate the relative proportions of haplo-X, diplo-X and triplo-X ova as 0.06:0.90:0.04, respectively, for LFM and 0.56: 0.44:0.00, respectively, for HFM. The data do not permit an estimate of the proportion of nullo-X ova. If we assume that these frequencies apply also to sperm, we can calculate the expected proportions of LFM, HFM and male selfprogeny of LFM and HFM animals. For the purpose of these calculations, we assume that 4A;5X is LFM. The calculated frequencies of LFM, HFM and male animals are 0.89, 0.11 and 0.004, respectively, for the self-progeny of an LFM animal and 0.20, 0.49 and 0.31, respectively, for the self-progeny of an HFM animal. These calculations agree fairly well with the tetraploid selfprogeny data of Table 1 (neglecting the unclassified progeny), although they neglect the contributions of nullo-X gametes. If the meiotic behavior of the X chromosome in tetraploids is indicative of autosomal segregations, then we would expect a relatively high proportion of aneuploid zygotes. By applying the frequency of 0.90 f o r diplo-X ova produced by LFM animals to each of the autosomes, for example, we calculate that only about 59% of ova will be diploid for all five autosomes. The sexual phenotype of D. melanogaster depends on the ratio of X chromosomes to autosomes (BRIDGES1939) ; thus, XX and X O diploids are females and 1925). (sterile) males, respectively, and 3A;2X flies are intersexes (BR~DGES The sexual phenotype of C . elegans also depends on the X-to-autosome ratio: 2A;2X7 3A;3X, 4A;4X and 4A;3X chromosome constitutions lead to hermaphrodite development, and 2A;IX, 3A;2X and 4A;2X give males. One can imagine a cellular mechanism for counting X chromosomes that involves a single dosesensitive site on the X: two copies of the site in a diploid would result in female development in the case of Drosophila-hermaphrodite development in the case of Caenorhabditis-and one copy would give a male; a duplication of the site could transform an otherwise XO animal into a female (or C. elegans hermaphroditej, and a deficiency of the site in a deficiency heterozygote would lead to male development. This single-site mechanism was shown by DOBZHANSKY and
400
J. E. MADL A N D R. K. HERMAN
SCHULTZ(1934) and PIPKIN(1940) not to apply to Drosophila. These workers looked at the sexual characteristics of animals carrying X-chromosome duplications or deficiencies of varying extents in addition to two complete X chromosomes and three sets of autosomes. It was concluded that at least several dosesensitive sites that act cumulatively are distributed throughout the Drosophila X chromosome-excluding the heterochromatic region. PIPKIN(1960) has also attempted to identify autosomal dose-sensitive sites affecting sex determination. Her studies on the sexual phenotypes of triploid aneuploids led to the suggestion that both the second and third chromosomes of Drosophila may be responsible for the shift toward maleness found in ordinary 3A;ZX intersexes. We note that the X-to-autosome ratio is not the only factor determining sexual phenotype in C . eleguns. Mutations in a number of genes have been identified that confer either male traits on 2A;ZX animals (KLASS,WOLFand HIRSH1976, and BRENNER1977; BEGUETand GIBERT1978) or hermaph1979; HODGKIN rodite traits on 2A;lX animals (NELSON, LEWand WARD 1978; HODGKIN, personal communication). Following the design of the Drosophila work, we have looked at the effect on the C. eleguns sexual phenotype of adding X-chromosome duplications in single dose to a ?A;2X chromosome constitution. The effectiveness of the X-chromosome duplications correlated with their apparent size (Figure 2). mnDp8 and mnDp27, which appear to be the smallest of the five duplications we have studied, had no discernable effect on the sexual phenotype of 3A;ZX animals. mnDp9 and mnDp25 were partially effective in shifting the sex of otherwise 3A;XZ animals from male to hermaphrodite; normal-appearing males and hermaphrodites as well as intersexes were produced. mnDpl0, which appears to be the largest of the five duplications, was clearly more effective than either mnDp9 o r mnDp25 in effecting the same conversion: virtually all ?A;2X animals carrying a single copy of mnDplO were hermaphrodites. On the other hand. 2A;lX animals carrying a single copy of mnDplO are fertile males; hence, mnDplO is not as effective as a complete X chromosome in determining hermaphroditism. The results lead to the conclusion that there exists on the C. eleguns X chromosome not one but at least three (and perhaps many more) dose-sensitive sites that act cumulatively in determining sex: mnDp9 and mnDplO must carry at least one site, mnDplO must have a second site and the X chromosome must have a third site. A question that we have not completely answered concerns the phenotype of XO diploids homozygous f o r the X-chromosome duplications. We have constructed four stocks. each homozygous for him-5(e14&7) V , unc-? X, and either mnDp8. mnDp9, mnDplO or mnDp25. him-5 is a recessive mutation that results HORVITZ and BRENNER in the production of 16% XO self-progeny (HODGKIN, 1979). Among these four stocks, only the one carrying mnDp8 gives identifiable male progeny (which tend to be smaller than normal). The failure to observe males in the other stocks may be due to inviability caused by genetic imbalance rather than shift in sex; nonetheless it is of interest that mnDp8, as we have already argued, appears to be the smallest duplication in this set of four. The variation in sexual phenotypes among 3A;ZX animals carrying a single
C. elegans POLYPLOIDS
40 1
copy of mnDp9 or mnDp25 may be due, at least in part, to slight variations in autosome numbers. On the other hand, seme of the variation may be analogous to what is seen with 3A;2X Drosophila: patches of male structures and patches of female structures in irregular patterns, even though all the cells have the same chromosome constitution (STERN1968). This analogy should apply at least to the variation in phenotype within each of the intersex animals we observed, since presumably the intersex phenotypes were not reflections of genotypic mosaicism. We have detected some germ line instability for some of our duplicaMADLand KARI1979), but the frequency of loss seems much tions (HERMAN, too low to account for the sexual mosaicism in the triploids; moreover, the duplication that gave the greatest frequency of loss, mnDplG, did not produce triploid intersexes. The production of C. elegans intersexes at a critical X-to-autosome ratio indicates that the assessment of this ratio is made by more than one cell during development and that different cells can differ in their assessments. Newly hatched male and hermaphrodite larvae have morphologically identical gonad primordia, each consisting of four cells, but the two sexes can be distinguished at this stage on the basis of the presence o r placement of certain somatic cells, both ectodermal and mesodermal (SULSTON and HORVITZ 1977). Numerous other somatic differences in muscle, hypodermis, nerve and other tail ectoderm also appear post-embryonically, and the lineages of these cells have been elucidated (SULSTON and HORVITZ 1977). Of course, post-embryonic gonad developOPPENHEIM and KLASS1976; KLASS, ment also differs in the two sexes (HIRSH, WOLFand HIRSH1976; J. KIMBLEand D. HIRSH,personal communication). Perhaps a careful analysis in triploid intersexes of the sexual phenotypes of sexspecific cells and their lineages would tell something about the nature of the assessments of the X-to-autosome ratio made during development. Indeed, it might prove useful to look for any effects of standard triploidy or tetraploidy alone on cell lineage patterns, both embryonic (DEPPEet al. 1978) and postembryonic (SULSTON and HORVITZ 1977). J. DUCKETT and R. RUSSELL have proposed (personal communication), on the basis of the measured activities in males and hermaphrodites of a species of acetylcholinesterase that appears to be encoded by an X-linked gene, that a dosage compensation mechanism may be operating in C. elegans. Measurements of X-linked gene activities in triploid males, females and intersexes of D . melanogaster have helped define the nature of dosage compensation in that organism (for review see LUCCHESI 1977). Analogous measurements for C. elegans might prove useful. We thank CLAIREKARIfor able technical help. This work was supparted by Public Health Service grant GM22387. LITERATURE CITED
BEGUET,B. and M.-A. GIBERT,1978 Obtention d'un mutant hermaphrodite autofkcond muni d'un bourse copulatrice mhle chez le Nematode libre Caenorhabditis elegans, lignke Bergerac. C. R. Acad. Sci. Pans, D, 286: 989-992.
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BRENNER,S., 1974 The genetics of Caenorhabditis elegans. Genetics 77: 71-94. BRIDGES,C. B., 1925 Sex in relation to chromosomes and genes. Am. Naturalist 59: 127-137. -, 1939 Cytological and genetic basis of sex. In: Sex and Internal Secretions, Second edition. Edited by E. ALLEN,C. H. DANFORTH and E. A. DOISY.Williams and Wilkens, Baltimore. DEPPE,V., E. SCHIERENBERG, T. COLE,C. KRIEG,D. SCHMITT, B. YODER and G. VON EHRENSTEIN, 1978 Cell lineages of the embryo of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S. 75: 376-380. T. and J. SCHULTZ,1934 The distribution of sex-factors in the X-chromosome DOBZHANSKY, of Drosophila melnnogaster. J. Genet. 28: 347-386. HERMAN, R. K., J. E. MADLand C. K. KARI, 1979 Duplications in Caenorhabditis elegans. Genetics 92: 419-435. HIRSH,D., D. OPPENHEIMand M. KLASS,1976 Development of the reproductive system of Caenorhabditis elegants. Develop. Biol. 49 : 200-219.
HODGKIN, J. A. and S. BRENNER,1977 Mutations causing transformation of sexual phenotype in the nematode Caenorhbditis elegans. Genetics 86: 275-287. HODGKIN, J., H. R. HORVITZ and S. BRENNER, 1979 Nondisjunction mutants of the nematode Caenorhabditis elegans. Genetics 91 : 67-94. KLASS,M., N. WOLFand D. HIRSH, 1976 Development of the male reproductive system and sexual transformation in the nematode Caenorhabditis elegans. Develop. Bid. 52 : 1-18.
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1979 Further characterization of a temperature-sensitive transformation mutant i n Caenorhabditis elegans. Develop. Biol. 69 : 329-335.
LUCCHESI,J. C., 1977 Dosage compensation: transcription-level regulation of X-linked genes in Drosophila. Am. Zoologist 17: 685-693. NELSON,G. A., K. K. LEW and S. WARD,1978 Intersex, a temperature-sensitive mutant of the nematode Caenorhabditis elegans. Develop. Bid. 66 : 386-409. NIGON,V., 1949a Effets de la polyploYdie chez un Ndmatode libre. C. R. Acad. Sci., Paris. 228: 1161-1162. -, 1949b Les modalit& de la reproduction et le dkterminisme de 1951a sexe chez quelques Nkmatodes libres. Ann. Sci. Nat. Zool. ser 11, 2: 1-132. -, La gamhtogknese d'un Nkmatode tetraploide obtenu par voie expkrimentale. Bull. Soc. Hist. Nat. Toulouse 86: 192-200. __ , 1951b Polyplo'idie exPCrimentale chez un Nkmatode libre, Rhabditis elegans Maupas. Bull. biol. Fr. Belg. 8 5 : 187-225.
PIPKIN, S. B., 1940 Multiple sex genes in the X-chromosome of Drosophila melanogaster. Univ. Texas Publ. 4032: 126-156. -, 1960 Sex balance in Drosophila melanogaster: aneuploidy of long regions of chromosome 3, using the triploid method. Genetics 45: 1205-1216.
STERN,C., 1968 Genetic Mosaics and Other Essays. Harvard University Press, Cambridge.
SULSTON, J. E. and H. R. HORVITZ, 1977 Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Develop. Biol. 5 6 : 110-156. Corresponding editor: A. CHOVNICK
Department of Genetics and Cell Biology, Unizjersity of Minnesota, Saint Paul, Minnesota 55108 Manuscript received June 22, 1979 ABSTRACT
Tetraploid stocks of Caenorhabditis elegans var. Bristol carrying autosomal and X-linked markers have been produced. Tetraploid hermaphrodites fall into two categories: those that give about 1% male self-progeny and those that give 25 to 40% male self-progeny, The former are basically 4A;4X-four sets of autosomes and four sex chromosomes-and the latter are 4A;3X. Males are 4A;ZX. (Diploid hermaphrodites are 2A;2X; males are 2A;IX.) Triploids wsre produced by crossing tetraploid hermaphrodites and diploid males. Triploids of composition 3A;3X are hermaphrodites; 3A;ZX animals are fertile males. Different X-chromosome duplications were added to a 3A;ZX chromosome constitution to increase the X-to-autosome ratio. Based on the resulting sexual phenotypes, we conclude that there exists on the C . elegans X chromosome at least three (and perhaps many mare) dose-sensitive sites that act cumulatively in determining sex.
A tetraploid stock of the small, free-living nematode, Caenorhclbditis elegans, was obtained some years ago by NIGON (1949a, 1951a, 1951b) from a heat-treated culture and maintained for 78 generations until accidentally lost. NIGON ( 1949a) reported that tetraploid hermaphrodites were larger than diploids and fell into two classes, which he called thklygknes and allklogches, based on the sex ratios of their self-progeny. Thklygknes gave about 0.7% male progeny and alldogknes gave about 40%. NIGONalso inspected Feulgenstained oocytes and spermatocytes from tetraploid animals and decided that th4ygknes were basically 4A;4X (four sets of autosomes and four sex chromosomes), alldogknes were 4A;3X, and males were 4A;2X. He also noted that many meiocytes showed irregular chromosome constitutions and suggested that the numerous sterile and weakly fertile animals he encountered may have been aneuploid (NIGON 1951b) . NIGON (1949b) had earlier shown that diploid hermaphrodites were 2A;2X and males were 2A;IX. I n the work reported here, we produced tetraploid lines of C. elegans var. Bristol carrying X-linked and autosomal visible markers (BRENNER 1974) and used these lines in crosses with diploid male stocks, some of which carried X-chromosome duplications, to investigate the relationship between chromosome composition and sex determination in C. elegans. Genetxs 93: 393402 October, 1979
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J. E. M A D L ARTD R. K. H E R M A N MATERIALS AND METHODS
The mutations and X-chromosome duplications used, the methods of culturing the animals and the fluorescence staining procedures have been described by BRENNER(1974) and HERMAN, MADLand KARI(1979). Tetraploid hermaphrodites whose progeny were to be enumerated were transferred daily to fresh plates. Tetraploid progeny to be classified by progeny testing were picked singly as larvae to be sure that no mating had occurred. Our original tetraploid line was derived as follows. Separate cultures of N2 (wild type) males and dpy-11 V; unc-3 X hermaphrodites, normally grown at 20”, were shifted to 27.5” for 12 hr and then mated. Wild-type hermaphrodite progeny were picked and screened for unusually low frequencies of Dpy and Unc phenotypes among their self-progeny. One line of very low fertility segregated males and hermaphrodites that seemed larger than normal and showed, by fluorescence staining, to hace a chromosome count higher than diploid-probably triploid originally. Continued selection for large animals of higher fertility eventually produced a quite stable tetraploid line. This stock continued to segregate Dpy and Unc animals, and we established homozygous tetraploid stocks derived from it: SP344: dpy-11; unc-3 X; SP345: dpy-11 V; SP346: wild-type; and SP347: unc-3 X. SP347 was mated with dpy-5 I / + diploid males, from which wild-type triploid hermaphrodites were picked. Continued selection for higher fertility of Dpy Unc segregants eventually resulted in the SP34.3 tetraploid stock, dpy-5 I ; unc-3 X . SP348, which is dpy-11 V ; unc-6X, was established i n analogous fashion using SP345 and mnDp30(X;f)/unc-6/0 diploid males to generate the triploid intermediate. A Zeiss Universal microscope was used for Nomarski differential interference microscopy. RESULTS
Tetraploids: I n agreement with NIGON( 1951b) we found that tetraploid hermaphrodites were of two types, based on the frequencies of male self-progeny. Those that gave 10% or less male self-progeny we called LFM (low frequency male producer), and those that gave more than 10% male self-progeny we called HFM. Animals with less than five progeny were not classified. Table 1 gives the frequencies of self-progeny from LFM and HFM animals. The data show that HFM animals segregated not only a large proportion of males but also large fractions of both HFM and LFM animals, whereas LFM hermaphrodites gave predominantly LFM progeny. The frequencies in Table 1 agree well with NIGON’S data (1951b), with the possible exception of the category termed “not classifiable.” NIGONnoted that many tetraploid animals were sterile or weakly fertile, but he did not quote frequencies for that class. Wild-type and dpy-22 V ; unc-3 X tetraploids gave similar self-progeny ratios from LFM and HFM animals, and the data from both have been combined in Table 1. The egg-hatching ~
TABLE 1
Self-progeny of tetraploid hermaphrodites Parent
LFM
Progeny frequencies HFM Male
LFM HFM
0.75 0.15
0.06 0.30
0.01 0.26
Not classifiable.
Number of progeny counted
0.18 0.29
936 701
* These were animals, some arrested as larvae, that gave less than five self-progeny.
C.elegans POLYPLOIDS
395
Irequency from LFM hermaphrodites was measured: 87% of 218 eggs hatched. The average brood size of ten dpy-12; unc-3 LFM hermaphrodites was 65 and of five wild-type LFM hermaphrodites was 84. Diploid hermaphrodites generally give about 300 animals per brood. W e stained oocytes and a few male spermatocytes with Hoechst 33258, with results that were consistent with NIGON'S cytological findings; for example, the oocyte shown in Figure lb, showing 12 bivalents, is typical of those found in the progeny of LFM animals, but we have also observed irregular patterns in which one or two extra chromosomes were present or one or two chromosomes appeared to be missing. Triploids: Triploid animals were produced by mating diploid N 2 males and tetraploid hermaphrodites bearing an autosomal dpy marker and an X-linked unc. The oocytes of wild-type hermaphrodite progeny generally showed, by fluorescence staining, 18 chromosomes, often appearing as six bivalents and six univalents, as shown in Figure lc. Although we have not made measurements, triploid animals are clearly larger than diploids. Of 3,864 eggs laid by triploid hermaphrodites, only 15% hatched. This frequency is still much higher than would be expected if only euploid zygotes hatched, and indeed, of 187 adult progeny of triploids, 32% were hermaphrodites that gave four or fewer offspring and 17% were males. Furthermore, oocytes of progeny from triploids were often seen to be aneuploid. The results of mating diploid males with tetraploid hermaphrodites depended on whether the hermaphrodites were LFM or HEM. I n the case of LFM hermaphrodites, we would expect most ova to be diplo-X, half of which would be fertilized by X-bearing sperm to give wild-type 3A;3X hermaphrodites, and half of which would be fertilized by nullo-X sperm to give 3A;2X Unc-3 animals. Since the vast majority of the Unc-3 animals issuing from this cross were in fact male (Table 2), we conclude that 3A;2X individuals are male. The rare Unc-3 hermaphrodites and wild-type males could be due to the occurrence of triplo-X and haplo-X ova, respectively. When HFM animals were crossed with diploid males, many wild-type male progeny were produced (Table 2), presumably as the result of the fusion of unc-3 haplo-X ova and unc-3+ haplo-X sperm. That at least some of these males were in fact unc-3/unc-3+ was shown by mating them with dpy-21 V; unc-3 X diploid hermaphrodites: non-Dpy Unc-3 hermaphrodite progeny, which must have received an unc-3 gene from the triploid male, were produced. This result also showed that triploid males can be fertile. Another point to be made from
FIGURE1 .--Fluorescence microscopy of oocytes stained with Hoechst 33058. (a) Diploid. (b) Tetraploid. (c) Triploid. Magnification in a-c: 1760x.
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TABLE 2
Cross progeny of wild-type diploid males and tetraploid hermphrodites Hermaphrodite parent'
dpy-5 I ; unc-3X LFM dpy-11 V ; unc-3 X LFM dpy-11 V ; unc-6X LFM d p y - l i V ; unc-3 X HFM
Frequencies of non-Dpy phenotypes Wild-type Wild-type Unc-3 Unc-3 hermaphrodite male hermaphrodite male
0.52 0.51
0.55 0.35
0.02 0.04 0.03 0.39
0.02 0.02 0.01 0.00
0.M 0.43 0.41 0.26
Number of progeny counted
901 1,139 641) 298
* Each mating plate contained a single hermaphrodite. The phenotype of H F M animals used in crosses was confirmed on the basis of the frequency of males among (Dpy Unc) self-progeny. For LFM crosses, the progeny of LFM animals were used, and their classification as LFM was confirmed on the basis of the frequency of wild-type male cross-progeny.
the data of Table 2 is that only 26% of the cross progeny of the HFM animals were Unc-3 (as compared with 45% from LFM animals), which suggests that many nullo-X sperm were lost to inviable zygotes. The simplest interpretation of this result is that 3A;lX zygotes are inviable. We have also generated triploids by crossing tetraploid males and diploid hermaphrodites. We would expect these triploids to be largely 3A;2X, and indeed, 98 % of the cross progeny (among 295 animals counted) were male. Triploids bearing X-chromosome duplications: If sex is determined in C. elegans, as it is in Drosophila, by the X-to-autosome ratio, then a ratio of 0.67 (in triploids) gives rise to a male and a ratio of 0.75 (in tetraploids) gives a hermaphrodite. (In normal diploid males and hermaphrodites, the X-to-autosome ratios are 0.5 and 1.0, respectively.) We were therefore prompted to construct animals with intermediate X-to-autosome ratios by adding X-chromosome duplications to a 3A;ZX chromosome constitution. We used five different X-chromosome duplications, all of which carry unc-3f and one or more neighboring loci MADLand KARI (Figure 2 ) and are translocated to LGZ or LGZZ (HERMAN, 1979). Each of the five duplications is homozygous viable in diploid hermaphrodites, suggestifig that very little of the autosomal linkage group to which each i s attached is missing. We conducted two sets of experiments: in both sets, males of genotype Dp/+; unc-3/0 were crossed with LF'M tetraploid hermaphrodites homozygous f o r an autosomal d p y marker and an X-linked unc marker. In one set of experiments (Table 3 ) , the unc marker was unc-3, and in the other (Table 4),unc-6, which is not covered by any of the duplications, was used. The relative frequencies of the four sperm genotypes were determined from control crosses using diploid d p y ; unc-3 hermaphrodites; that is, in these crosses Dp/X, Dp/O, X and nullo-X sperm produced wild-type hermaphrodites, wild-type males, Unc non-Dpy hermaphrodites, and Unc non-Dpy males, respectively. The results of the crosses with diploid hermaphrodites are given in Table 3 and suggest a tendency for the Dp-bearing chromosome, particularly in the case of mnDpl0, to segregate from the X during meiosis in males (HERMAN. MADLand KARI 1979). In any case, if we compare the triploid progeny ratios in Table 3 with the diploids, we see that in three cases the frequency of wild-type triploid hermaphrodites increases
C . elegans POLYPLOIDS
---
dpy-6
~ n c - 8 4~ c - 3 unc-7
UnC-9
I
I
I
I
397
I
I
I
I
I
mnDp8 ( X ; I 1
mnDp9 ( X ; Z )
mnDp70 ( X ; l )
-
I
I
mnDp25 (41)
P
mnDp27
(&n1
FIGURE 2.-Genetic extents of the X-chromosome duplications used in this work (HERMAN, MADLand KARI 1979). TABLE 3 Cross progeny of X-duplication-bearingdiploid males and diploid or LFM tetraploid hermaphrodites
Duplication*
Ploidy of hermaphrodite+
mnDp8 mnDp8 mnDp9 mnDp9 mnDplO mnDplO mnDp25 mnDp25 mnDp27 mnDp27
diploid tetraploid diploid tetraploid diploid tetraploid diploid tetraploid diploid tetraploid
Frequencies of non-Dpv phenotypes Unc-3 Wild-type Wild-type Unc-3 male hermauhrudite male hermauhrodite
0.27 0.27 0.27 0.09 0.27 0.02 0.25 0.12 0.27 0.20
0.25 0.27 0.21 0.39 0.21 0.45 0.24 0.38 0.26 0.28
0.27 0.25 0.28 0.28 0.33 0.32 0.28 0.29 0.26 0.31
0.22 0.21 0.23 0.24 0.20 0.20 0.23 0.21 0.22
0.u)
Number of progeny counted
1,017 272
605 658 357 908 813 536 75 1 836
* Males all had the following genotype: Dp/+; unc-3/0.
tHermaphrodites were all homozygous for an
and unc-3 X .
autosomal dpy marker (dpy-5 I or dpy-11 V )
TABLE 4 Cross-progeny of Dp/+; unc-3/0 diploid males and dpy-I 1 V; unc-6 X LFM tetraploids
Duplication
mnDp8 mnDp9 mnDplO mnDp25 mnDp27
Frequencies of non-Dpy phenotypes Wild-type Wild-type Unc-6 hermaphrodite male hermaphrodite
0.56 0.53 0.53 0.56 0.54
0.04 0.02 0.03 0.05 0.03
0.02 0.17 0.29 0.12 0.02
Unc-6 male
Number of progeny counted
0.38 0.28 0.16 0.28 0.41
518 389 280 520 288
398
J . E. MADL AND R. K. HERMAN
at the expense of wild-type triploid males. This effect was most extreme in the case of mnDpl0, where very few wild-type males were produced, but it is also clear with mnDp9 and mnDp25. We conclude that adding mnDpZO to 3A;2X generally (if not always) gives a hermaphrodite, whereas adding mnDp9 or mnDp25 sometimes gives a hermaphrodite and sometimes a male. Indeed, in both the mnDp9 and mnDp25 crosses, about 10 to 20% of the animals classified in Table 3 as males, on the basis of male-like tails, proved upon closer inspection by Nomarski microscopy to be obvious intersexes, showing besides some development of a male-like tail, some oogenesis and sometimes even fertilized eggs and a vestigial vulva (Figure 3). Adding mnDp8 or mnDp27 to 3A;2X appeared to permit normal male development. Wild-type males appeared at about the same frequency as in diploid crosses, and intersexes were not common: 67 wild-type males and 67 non-Dpy Unc-3 males from a mnDp8 cross were inspected by Nomarski microscopy and no indication of intersexuality was found. As expected, the sex ratios for Unc progeny (which did not contain duplications) in every case were the same in triploids versus diploids in Table 3. I n the second set of experiments, in which the D p / + ; unc-3 X / O diploid males were crossed with individual dpy-11 V; unc-6 X LFM animals, the 3A;2X animals were represented by the non-Dpy Unc-6 progeny, roughly half of which (or more, particularly in the case of mnDpZ0; see diploid results in Table 3) should carry the duplication. If a duplication converts 3A;2X males to hermaphrodites, then the frequency of non-Dpy Unc-6 hermaphrodites, which is about 0.02 in the absence of a duplication (Table 2). should increase at the expense of the non-Dpy Unc-6 male class. Furthermore, the sex ratio of non-Dpy non-Unc animals (which are 3A;3X) should be unaffected by the duplications. These predictions were satisfied by the results, given in Table 4. Over half of the non-
FIGURE3.-Nomarski microscopy of three intersex aninials. (a) 3A;2X/mnDp25 with a male tail and oocytes. Magnification: 1 0 0 ~ (b) . 3A;2X/mnDp25 with a male tail and fertilized eggs. Magnification: 1 9 0 ~ .(c) 3A;2X/mnDp9 with a partially developed male tail and fertilized eggs. Magnification: 2 3 0 ~ .
C. elegans POLYPLOIDS
399
Dpy Unc-6 progeny in the case of the mnDplO cross were hermaphrodites. As in the first set of experiments, mnDp9 and mnDp25 appeared to be partially effective in converting 3A;2X animals to hermaphrodites, and, as before, many of the classified male progeny proved upon closer inspecticln to be intersexes. Also as before,nnDp8 and mnDp27 appeared to be ineffective in converting 3A;2X males to hermaphrodites. DISCUSSION
We can use the data of Table 2 to estimate the relative proportions of haplo-Xi, diplo-X and triplo-X ova produced by LFM (4A;4X) and HFM (4A;3X) animals, on the basis of the following assumptions: diploid males produce equal proportions of haplo-X and nullo-X sperm, 3A;jX is inviable, 3A;2X is male, and 3A;3X and 3A;4X are viable hermaphrodites. The rationale for assuming the viability of 3A;4X is simply that 2A;3X animals are viable (HODGKIN, HORVITZ and BRENNER 1979), but the calculations are very insensitive to this assumption in any case. We calculate the relative proportions of haplo-X, diplo-X and triplo-X ova as 0.06:0.90:0.04, respectively, for LFM and 0.56: 0.44:0.00, respectively, for HFM. The data do not permit an estimate of the proportion of nullo-X ova. If we assume that these frequencies apply also to sperm, we can calculate the expected proportions of LFM, HFM and male selfprogeny of LFM and HFM animals. For the purpose of these calculations, we assume that 4A;5X is LFM. The calculated frequencies of LFM, HFM and male animals are 0.89, 0.11 and 0.004, respectively, for the self-progeny of an LFM animal and 0.20, 0.49 and 0.31, respectively, for the self-progeny of an HFM animal. These calculations agree fairly well with the tetraploid selfprogeny data of Table 1 (neglecting the unclassified progeny), although they neglect the contributions of nullo-X gametes. If the meiotic behavior of the X chromosome in tetraploids is indicative of autosomal segregations, then we would expect a relatively high proportion of aneuploid zygotes. By applying the frequency of 0.90 f o r diplo-X ova produced by LFM animals to each of the autosomes, for example, we calculate that only about 59% of ova will be diploid for all five autosomes. The sexual phenotype of D. melanogaster depends on the ratio of X chromosomes to autosomes (BRIDGES1939) ; thus, XX and X O diploids are females and 1925). (sterile) males, respectively, and 3A;2X flies are intersexes (BR~DGES The sexual phenotype of C . elegans also depends on the X-to-autosome ratio: 2A;2X7 3A;3X, 4A;4X and 4A;3X chromosome constitutions lead to hermaphrodite development, and 2A;IX, 3A;2X and 4A;2X give males. One can imagine a cellular mechanism for counting X chromosomes that involves a single dosesensitive site on the X: two copies of the site in a diploid would result in female development in the case of Drosophila-hermaphrodite development in the case of Caenorhabditis-and one copy would give a male; a duplication of the site could transform an otherwise XO animal into a female (or C. elegans hermaphroditej, and a deficiency of the site in a deficiency heterozygote would lead to male development. This single-site mechanism was shown by DOBZHANSKY and
400
J. E. MADL A N D R. K. HERMAN
SCHULTZ(1934) and PIPKIN(1940) not to apply to Drosophila. These workers looked at the sexual characteristics of animals carrying X-chromosome duplications or deficiencies of varying extents in addition to two complete X chromosomes and three sets of autosomes. It was concluded that at least several dosesensitive sites that act cumulatively are distributed throughout the Drosophila X chromosome-excluding the heterochromatic region. PIPKIN(1960) has also attempted to identify autosomal dose-sensitive sites affecting sex determination. Her studies on the sexual phenotypes of triploid aneuploids led to the suggestion that both the second and third chromosomes of Drosophila may be responsible for the shift toward maleness found in ordinary 3A;ZX intersexes. We note that the X-to-autosome ratio is not the only factor determining sexual phenotype in C . eleguns. Mutations in a number of genes have been identified that confer either male traits on 2A;ZX animals (KLASS,WOLFand HIRSH1976, and BRENNER1977; BEGUETand GIBERT1978) or hermaph1979; HODGKIN rodite traits on 2A;lX animals (NELSON, LEWand WARD 1978; HODGKIN, personal communication). Following the design of the Drosophila work, we have looked at the effect on the C. eleguns sexual phenotype of adding X-chromosome duplications in single dose to a ?A;2X chromosome constitution. The effectiveness of the X-chromosome duplications correlated with their apparent size (Figure 2). mnDp8 and mnDp27, which appear to be the smallest of the five duplications we have studied, had no discernable effect on the sexual phenotype of 3A;ZX animals. mnDp9 and mnDp25 were partially effective in shifting the sex of otherwise 3A;XZ animals from male to hermaphrodite; normal-appearing males and hermaphrodites as well as intersexes were produced. mnDpl0, which appears to be the largest of the five duplications, was clearly more effective than either mnDp9 o r mnDp25 in effecting the same conversion: virtually all ?A;2X animals carrying a single copy of mnDplO were hermaphrodites. On the other hand. 2A;lX animals carrying a single copy of mnDplO are fertile males; hence, mnDplO is not as effective as a complete X chromosome in determining hermaphroditism. The results lead to the conclusion that there exists on the C. eleguns X chromosome not one but at least three (and perhaps many more) dose-sensitive sites that act cumulatively in determining sex: mnDp9 and mnDplO must carry at least one site, mnDplO must have a second site and the X chromosome must have a third site. A question that we have not completely answered concerns the phenotype of XO diploids homozygous f o r the X-chromosome duplications. We have constructed four stocks. each homozygous for him-5(e14&7) V , unc-? X, and either mnDp8. mnDp9, mnDplO or mnDp25. him-5 is a recessive mutation that results HORVITZ and BRENNER in the production of 16% XO self-progeny (HODGKIN, 1979). Among these four stocks, only the one carrying mnDp8 gives identifiable male progeny (which tend to be smaller than normal). The failure to observe males in the other stocks may be due to inviability caused by genetic imbalance rather than shift in sex; nonetheless it is of interest that mnDp8, as we have already argued, appears to be the smallest duplication in this set of four. The variation in sexual phenotypes among 3A;ZX animals carrying a single
C. elegans POLYPLOIDS
40 1
copy of mnDp9 or mnDp25 may be due, at least in part, to slight variations in autosome numbers. On the other hand, seme of the variation may be analogous to what is seen with 3A;2X Drosophila: patches of male structures and patches of female structures in irregular patterns, even though all the cells have the same chromosome constitution (STERN1968). This analogy should apply at least to the variation in phenotype within each of the intersex animals we observed, since presumably the intersex phenotypes were not reflections of genotypic mosaicism. We have detected some germ line instability for some of our duplicaMADLand KARI1979), but the frequency of loss seems much tions (HERMAN, too low to account for the sexual mosaicism in the triploids; moreover, the duplication that gave the greatest frequency of loss, mnDplG, did not produce triploid intersexes. The production of C. elegans intersexes at a critical X-to-autosome ratio indicates that the assessment of this ratio is made by more than one cell during development and that different cells can differ in their assessments. Newly hatched male and hermaphrodite larvae have morphologically identical gonad primordia, each consisting of four cells, but the two sexes can be distinguished at this stage on the basis of the presence o r placement of certain somatic cells, both ectodermal and mesodermal (SULSTON and HORVITZ 1977). Numerous other somatic differences in muscle, hypodermis, nerve and other tail ectoderm also appear post-embryonically, and the lineages of these cells have been elucidated (SULSTON and HORVITZ 1977). Of course, post-embryonic gonad developOPPENHEIM and KLASS1976; KLASS, ment also differs in the two sexes (HIRSH, WOLFand HIRSH1976; J. KIMBLEand D. HIRSH,personal communication). Perhaps a careful analysis in triploid intersexes of the sexual phenotypes of sexspecific cells and their lineages would tell something about the nature of the assessments of the X-to-autosome ratio made during development. Indeed, it might prove useful to look for any effects of standard triploidy or tetraploidy alone on cell lineage patterns, both embryonic (DEPPEet al. 1978) and postembryonic (SULSTON and HORVITZ 1977). J. DUCKETT and R. RUSSELL have proposed (personal communication), on the basis of the measured activities in males and hermaphrodites of a species of acetylcholinesterase that appears to be encoded by an X-linked gene, that a dosage compensation mechanism may be operating in C. elegans. Measurements of X-linked gene activities in triploid males, females and intersexes of D . melanogaster have helped define the nature of dosage compensation in that organism (for review see LUCCHESI 1977). Analogous measurements for C. elegans might prove useful. We thank CLAIREKARIfor able technical help. This work was supparted by Public Health Service grant GM22387. LITERATURE CITED
BEGUET,B. and M.-A. GIBERT,1978 Obtention d'un mutant hermaphrodite autofkcond muni d'un bourse copulatrice mhle chez le Nematode libre Caenorhabditis elegans, lignke Bergerac. C. R. Acad. Sci. Pans, D, 286: 989-992.
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BRENNER,S., 1974 The genetics of Caenorhabditis elegans. Genetics 77: 71-94. BRIDGES,C. B., 1925 Sex in relation to chromosomes and genes. Am. Naturalist 59: 127-137. -, 1939 Cytological and genetic basis of sex. In: Sex and Internal Secretions, Second edition. Edited by E. ALLEN,C. H. DANFORTH and E. A. DOISY.Williams and Wilkens, Baltimore. DEPPE,V., E. SCHIERENBERG, T. COLE,C. KRIEG,D. SCHMITT, B. YODER and G. VON EHRENSTEIN, 1978 Cell lineages of the embryo of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S. 75: 376-380. T. and J. SCHULTZ,1934 The distribution of sex-factors in the X-chromosome DOBZHANSKY, of Drosophila melnnogaster. J. Genet. 28: 347-386. HERMAN, R. K., J. E. MADLand C. K. KARI, 1979 Duplications in Caenorhabditis elegans. Genetics 92: 419-435. HIRSH,D., D. OPPENHEIMand M. KLASS,1976 Development of the reproductive system of Caenorhabditis elegants. Develop. Biol. 49 : 200-219.
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1979 Further characterization of a temperature-sensitive transformation mutant i n Caenorhabditis elegans. Develop. Biol. 69 : 329-335.
LUCCHESI,J. C., 1977 Dosage compensation: transcription-level regulation of X-linked genes in Drosophila. Am. Zoologist 17: 685-693. NELSON,G. A., K. K. LEW and S. WARD,1978 Intersex, a temperature-sensitive mutant of the nematode Caenorhabditis elegans. Develop. Bid. 66 : 386-409. NIGON,V., 1949a Effets de la polyploYdie chez un Ndmatode libre. C. R. Acad. Sci., Paris. 228: 1161-1162. -, 1949b Les modalit& de la reproduction et le dkterminisme de 1951a sexe chez quelques Nkmatodes libres. Ann. Sci. Nat. Zool. ser 11, 2: 1-132. -, La gamhtogknese d'un Nkmatode tetraploide obtenu par voie expkrimentale. Bull. Soc. Hist. Nat. Toulouse 86: 192-200. __ , 1951b Polyplo'idie exPCrimentale chez un Nkmatode libre, Rhabditis elegans Maupas. Bull. biol. Fr. Belg. 8 5 : 187-225.
PIPKIN, S. B., 1940 Multiple sex genes in the X-chromosome of Drosophila melanogaster. Univ. Texas Publ. 4032: 126-156. -, 1960 Sex balance in Drosophila melanogaster: aneuploidy of long regions of chromosome 3, using the triploid method. Genetics 45: 1205-1216.
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SULSTON, J. E. and H. R. HORVITZ, 1977 Post-embryonic cell lineages of the nematode Caenorhabditis elegans. Develop. Biol. 5 6 : 110-156. Corresponding editor: A. CHOVNICK
